The long-term goal of this work is to improve outcomes in Ewing Sarcoma, the most lethal bone tumor of children. Ewing Sarcoma Family Tumors (ESFT) are the second most common bone and soft tissue cancer afflicting children, adolescents, and young adults. ESFT are characterized by a chromosomal translocation resulting in a fusion between EWS and a member of the ETS family of transcription factors, most commonly FLI1. ESFT are treated with intensive chemotherapy, radiation, and surgery, yet more than half of patients die of disease within five years of diagnosis, while survivors often suffer long-term deleterious effects of treatment. An immediate need exists for the development of effective and targeted therapies with decreased toxicity. Unfortunately, we still have a limited understanding of the molecular mechanisms of ESFT tumorigenesis, which has greatly impeded the identification of improved therapies. Our preliminary data indicate that recurrent areas of chromosomal gain and loss occur in Ewing Sarcoma, suggesting that amplification or deletion of cooperating genes in these recurring regions facilitate cell transformation by EWS-FLI1. Identification and validation of these cooperating genes is essential for the development of new therapies, because strategies to target EWS-FLI1 itself have not to date been successful. We previously developed a zebrafish model of ESFT that recapitulates key features of the human disease. We will now use the fish model for high-throughput functional genomic assays to validate promising candidate EWS-FLI1 effector genes emerging from our high- resolution genomic analyses of human ESFT. We will further capitalize on this assay by conducting small- molecule screens to identify lead compounds that act as inhibitors of EWS-FLI1-mediated cellular transformation. To achieve this goal we will 1) identify critical EWS-FLI1 effectors though high-resolution genomic analysis of tumors; 2) use zebrafish in vivo models of EWS-FLI1 activity to validate candidate ESFT effectors identified in genomic copy number analyses; and 3) identify small molecules capable of inhibiting EWS-FLI1 function in the zebrafish model At the completion of this study, we will have demonstrated the effectiveness of a novel genomic pipeline for discovery and functional analysis of cooperating genes in translocation positive sarcomas. This novel pipeline will take advantage of our combined expertise in copy number analysis and comparative oncology using zebrafish models. This new pipeline and its novel approach will lead to the rapid testing and introduction of new therapeutic agents for sarcomas, one of the most deadly types of cancer in children and adults.